The coagulation of blood is a complex process including the sequential interaction of a number of components, in particular of fibrinogen, factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI and factor XII. The loss of one of these components or the inhibition of its functionality leads to an increased tendency of hemorrhaging which may be life-threatening for some patients.
Von Willebrand factor (vWF) circulates in plasma complexed with factor VIII, factor VIII aiding the coagulation of blood and vWF in the complex with factor VIII stabilizing the latter and protecting it from proteolytic degradation. By its function in platelet aggregation, vWF also directly interferes in the coagulation of blood. vWF is a glycoprotein formed in different mammalian cells and subsequently released into circulation. Starting from a polypeptide chain having a molecular weight of approximately 220 kD, a vWF dimer having a molecular weight of 550 kD is formed in the cells by formation of several sulfur bonds. From the vWF dimers, further polymers of vWF of ever increasing molecular weights, up to 20 million Daltons, are formed by linkage. Therefore, vWF exists in plasma in a series of multimer forms having molecular weights of from 1×106 to 20×106 Daltons. It is assumed that particularly the high-molecular vWF multimers are of essential importance in the coagulation of blood.
Besides the carrier function for coagulation factor VIII, vWF has the functions of bridge formation between vessel wall and the platelets and of platelet agglutination. The basis for platelet agglutination is given by the binding of vWF to surface receptors (glycoproteins Ib, IIb/IIIa). The binding site within vWF for binding to GP Ib is located in disulfide loop Cys(509)-Cys(695). It is known that platelet agglutination starts with the binding of vWF to glycoprotein Ib. Following an activation signal, binding of vWF to the glycoprotein IIb/IIIa-complex and agglutination occur. Binding of vWF to the surface receptors thus is a prerequisite for platelet agglutination; the binding of several platelets by a vWF molecule leads to agglutination. vWF-platelet binding thus constitutes the molecular cause for platelet agglutination.
In hemophilia, blood coagulation is disturbed by a deficiency of certain plasmatic blood coagulation factors. In hemophilia A, the tendency to hemorrhage is based on a deficiency of factor VIII or on a deficiency of vWF, respectively, which is an essential component of factor VIII. Treatment of hemophila A primarily is effected by replacing the lacking coagulation factor by factor concentrates, e.g. by infusion of factor VIII, factor VIII-complex or vWF.
vWF Syndrome has several clinical pictures which go back to an underproduction or overproduction of vWF. Thus, e.g., an overproduction of vWF leads to an increased thrombosis tendency, whereas an undersupply is caused by the absence or reduction of high-molecular forms of vWF which manifests itself by an increased hemorrhage tendency and an extended hemorrhaging period due to an inhibited platelet aggregation. The deficiency of vWF may also cause a phenotypical hemophila A, since vWF is an essential component of functional factor VIII. In these instances, the half-life of factor VIII is reduced so much that its function in the blood coagulation cascade is impaired. Patients suffering from von Willebrand disease (vWD) thus frequently exhibit a factor VIII deficiency. In these patients, the reduced factor VIII activity is not the consequence of a defect of the X-chromosomal gene, but is an indirect consequence of the quantitative and qualitative change of vWF in plasma. The differentiation between hemophilia A and vWF normally can be effected by measuring vWF antigen or by determining the ristocetin cofactor activity. Both, the vWF antigen content and the ristocetin cofactor activity is lowered in most vWD patients, whereas it is normal in hemophilia A patients.
Conventional methods for the therapy of von Willebrand syndrome are with vWF recovered from plasma, and there exists a number of suggestions to treat vWD patients with purified vWF or with factor VIII/vWF-complex.
Purification of factor VIII or of factor VIII-complex from plasma or from cryoprecipitate is even more difficult because factor VIII is present in plasma in very small amounts only, is extremely unstable, and the association of factor VIII with vWF is reversible under specific conditions. Factor VIII is recovered from plasma by purification and concentration, yet, depending on the purification method, instability and loss of factor VIII activity may occur because vWF and factor VIII are separated during purification. Thus, the final product frequently is a mixture of stable factor VIII-complex and unstable factor VIII, as well as of-contaminating proteins, such as, e.g., fibrinogen, fibronectin or vitamin K-dependent proteins which could not be removed by the purification. Because of the instability of the purified complex, stabilizers, such as albumin or amino acids etc., are admixed. However, the presence of contaminating proteins and/or stabilizers in the purified product did reduce the specific activity of the factor VIII-complex.
EP 0 468 181 describes a method of purifying factor VIII from human plasma by ion exchange chromatography, elution of factor VIII with high ionic strength at acidic pH and collecting the eluate in the presence of a stabilizer, such as heparin, albumin and PEG and lysine or histidine as antiproteases. However, upon the addition of albumin, the specific activity decreases from 300-1200 U/mg protein to 18-24 U/mg protein.
Madaras et al. (Haemostasis 7:321-331 (1978) describe a method of purifying factor VIII on heparin-Sepharose and eluting with increasing NaCl concentrations. However, the factor VIII thus obtained had merely low activity.
U.S. Pat. No. 5,252,709 describes a method of separating factor VIII, vWF, fibronectin and fibrinogen from human plasma, wherein at first factor VIII, vWF and fibronectin are bound to a DEAE-type ion exchanger and subsequently are eluted separately from the ion exchanger by increasing salt concentrations.
Zimmerman et al. (U.S. Pat. No. 4,361,509) have described a method of purifying factor VIII, wherein factor VIII/vWF-complex is bound to a monoclonal anti-vWF antibody and factor VIII is dissociated from the complex by means of CaCl2 ions. The factor VIII thus obtained subsequently is recovered in pure form via a further chromatographic step, it must, however, be stabilized by the addition of human albumin.
By expressing factor VIII in recombinant cells (Wood et al. (1984), Nature 312:330-337), factor VIII could be produced by genetic engineering methods, yet only by the addition of or co-expression with vWF, a commercially usable yield of recombinant factor VIII could be obtained. To produce a pharmaceutical preparation, however, vWF is separated from factor VIII during the purification process up to a negligible residual amount, and the purified recombinant factor VIII is stabilized with albumin (Griffith et al. (1991), Ann. Hematol. 63:166-171).
For a use in the therapy of patients suffering from hemophilia A—and also from von Willebrand syndrome, a purified factor VIII, complexed with vWF, is desirable (Berntorp (1994), Haemostasis 24:289-297). In particular, it has repeatedly been emphasized that in preparations lacking vWF or having merely a low content of vWF, an extended bleeding time and a low factor VIII:C half-life are observed in vivo. Normalisation of vWF in vivo is important for maintaining the concentration of factor VIII in plasma both by reducing the factor VIII elimination rate and by aiding the liberation of endogenous factor VIII (Lethagen et al. (1992), Ann. Hematol. 65:253-259).
DE 3 504 385 describes carrying out an ion exchange chromatography for purifying factor VIII/vWF-complex, factor VIII-complex being bound via sulfate groups and eluted with citrated buffer, calcium chloride and NaCl gradient. In this instance, the factor VIII-complex is eluted from the carrier with a concentration of 0.5 M NaCl.
EP 0 416 983 describes the recovery of factor VIII/vWF-complex from human plasma by precipitation with a combination of barium chloride and aluminum hydroxide and subsequent anion exchange chromatography on DEAE-Fractogel.
In EP 0 411 810, purification of factor VIII/vWF-complex from cryoprecipitate is effected by means of heparin affinity chromatography and subsequent elution of the complex with calcium chloride. A further development of this method is described in WO 93/22337. To remove contaminating proteins, such as fibrinogen and fibronectin, a glycine/NaCl precipitation is carried out after the elution with CaCl2. To purify factor VIII/vWF-complex it has also been suggested to precipitate contaminating proteins, such as fibrinogen, with high concentrations of amino acids, in particular glycine, to dissociate factor VIII/vWF-complex which remains in solution by the addition of a calcium and amino acid-containing buffer, and subsequently to recover factor VIII and vWF separately from each other by anion exchange chromatography (WO 82/04395).
U.S. Pat. No. 5,356,878 describes the preparation of factor VIII-complex, in which contaminating proteins (fibrinogen, vitamin K-dependent factors or fibronectin) are separated by precipitation with Al(OH)3 and PEG, factor VIII-complex is chemically virus-inactivated in the presence of glycine and NaCl, and subsequently the non-factor VIII-complex-specific proteins are removed by gel filtration.
Hornsey et al. (Thromb. Haemost. 57:102-105 (1987)) have purified factor VIII/vWF by means of immune affinity chromatography and attained a specific activity of 45 U of factor VIII/mg protein and 60 U of ristocetin activity/mg protein. However, the final product is contaminated with 4% of fibrinogen and with 2% of fibronectin and with murine antibodies detached from the carrier.
Mejan et al. (Thromb. Haemost. 59:364-371 (1988)) suggested to purify factor VIII/vWF-complex directly from plasma by immune affinity chromatography. The purified complex was stabilized with human serum albumin and subsequently lyophilized. With the elution conditions described, however, a partial liberation of the antibodies from the column was observed, which led to a contamination of the eluate with monoclonal antibodies and required a second purification step for removal of the antibodies. With their method, Mejan et al. attained an approximately 1400-fold enrichment of the factor VIII/vWF-complex with a specific factor VIII:C-activity and ristocetin activity of 20 U/mg protein each, the product containing all the vWF multimers. After stabilization of the complex with 10 mg/ml albumin, a stability of 3-4 months at −20° C. was observed. However, it has been repeatedly emphasized that a particular difficulty in the purification of the complex consists in maintaining the association of the proteins, because both components in the complex are unstable.
Harrison et al. (Thromb. Res. 50:295-304 (1988)) describe the purification of factor VIII/vWF-complex by means of chromatography on dextran sulfate-agarose.
EP 0 600 480 describes the purification of factor VIII/vWF-complex by means of anion exchange chromatography, wherein the factor VIII/vWF-complex-containing eluate is stabilized with heparin and albumin and optionally lysine and histidine are added as antiproteases. Commercially available factor VIII/vWF-preparations partially have no or only a small portion of high-molecular vWF multimers (vWF/HMW), and exhibit, particularly in dependence on the infusion time, in vivo a reduction of the high-molecular vWF multimers (Lethagen et al. (1992), Ann. Hematol. 65:253-259).
The factor VIII preparations described in the prior art do mostly contain the entire vWF multimer pattern, yet their portions of HMW-vWF and LMW-vWF vary and they exhibit so-called triplet structures, indicating a proteolytic degradation of vWF multimers, in particular of vWF/HMW (Scott et al. (1993), Sem. Thromb. Hemost. 19:37-47, Baillod et al. (1992), Thromb. Res. 66:745-755, Mannucci et al. (1992), Blood 79:3130-3137). The stability of these preparations is limited thereby.
To stabilize the preparations, either before virus inactivation or so as to obtain a storage-stable preparation, it has repeatedly been emphasized that the addition of a stabilizer, such as albumin, is required.
All the factor VIII concentrates that have been obtained by purification of the protein from human plasma or which have been in contact with biological material from mammals furthermore bear the potential risk of containing microbiological or molecular pathogens, such as, e.g., viruses. To produce a safe preparation therefore an inactivation of pathogenic organisms is also always necessary. Effective inactivation methods may easily also lead to a loss of the biologic activity of the factor VIII complex. Thus, Palmer et al. found (Thromb. Haemost. 63:392-402 (1990)) that in case of heat treatment for an effective virus inactivation, an activity loss of between 17% and 30% must be reckoned with also in the presence of a stabilizer.
It has repeatedly been emphasized that factor VIII/vWF-concentrates exhibiting an intact multimer structure possibly have a favorable influence on the hemorrhaging time, because they carry out the primary function of vWF, i.e. platelet agglutination, and have a higher affinity to the platelet receptors glycoprotein Ib and IIb/IIIa than low-molecular vWF multimers (LMW-vWF) (Mannucci et al. (1987), Americ. J. Hematology 25:55-65). However, there exists the problem that there occurs a degradation particularly of the HMW-vWF molecules during the process of preparing factor VIII concentrates.
Thus, there is a need for a factor VIII-complex having a sufficient specific activity of factor VIII:C and vWF-activity, which has an improved stability and which remains stable over an extended period of time also without the addition of the non-factor VIII/vWF-complex-specific stabilizer.